Multilocus sequence typing schemes for the emerging swine pathogen Mycoplasma hyosynoviae.

Mycoplasma (M.) hyosynoviae is a commensal of the upper respiratory tract in swine, which has the potential to spread systemically, usually resulting in arthritis in fattening pigs and gilts. To date, very little is known about the epidemiology of M. hyosynoviae, mainly due to a lack of suitable typing methods. Therefore, this study aimed to develop both a conventional multi locus sequence typing (MLST) and a core genome (cg) MLST scheme. The development of the cgMLST was based on whole genome sequences of 64 strains isolated from pigs and wild boars during routine diagnostics as well as nine publicly available genomes. A cgMLST scheme containing 390 target genes was established using the Ridom© SeqSphere+ software. Using this scheme as a foundation, seven housekeeping genes were selected for conventional MLST based on their capability to reflect genome wide relatedness and subsequently, all 73 strains were typed by applying both methods. Core genome MLST results revealed a high diversity of the studied strain population and less than 100 allele differences between epidemiologically unrelated strains were only detected for four isolates from the US. On the other hand, seven clonal clusters (≤ 12 allele differences) comprising 20 isolates were identified. Comparison of the two typing methods resulted in highly congruent phylogenetic trees and an Adjusted Rand Coefficient of 0.893, while cgMLST showed marginally higher resolution when comparing closely related isolates, indicated by a slightly higher Simpson's ID (0.992) than conventional MLST (Simpson's ID = 0.990). Overall, both methods seem well suited for epidemiological analyses for scientific as well as diagnostic purposes. While MLST is faster and cheaper, cgMLST can be used to further differentiate closely related isolates.

Dynamics of antibody response and bacterial shedding of Mycoplasma hyorhinis and M. hyosynoviae in oral fluids from experimentally inoculated pigs.

Mycoplasma hyorhinis (Mhr) and M. hyosynoviae (Mhs) are commensal organisms of the upper respiratory tract and tonsils but may also cause arthritis in pigs. In this study, 8-week-old cesarean-derived colostrum-deprived (CDCD) pigs (n = 30; 3 groups, 10 pigs per group, 2 pigs per pen) were inoculated with Mhr, Mhs, or mock-inoculated with culture medium and then pen-based oral fluids were collected at different time points over the 56 days of the experimental study. Oral fluids tested by Mhr and Mhs quantitative real-time PCRs revealed Mhr DNA between day post inoculation (DPI) 5-52 and Mhs DNA between DPI 5-15. Oral fluids were likewise tested for antibody using isotype-specific (IgG, IgA, IgM) indirect ELISAs based on a recombinant chimeric polypeptide of variable lipoproteins (A-G) for Mhr and Tween 20-extracted surface proteins for Mhs. Mhr IgA was detected at DPI 7 and, relative to the control group, significant (p < 0.05) antibody responses were detected in the Mhr group between DPI 12-15 for IgM and DPI 36-56 for both IgA and IgG. In the Mhs group, IgM was detected at DPI 10 and significant (p < 0.05) IgG and IgA responses were detected at DPI 32-56 and DPI 44-56, respectively. This study demonstrated that oral fluid could serve as an effective and convenient antemortem sample for monitoring Mhr and Mhs in swine populations.

Detection and disease diagnosis trends (2017-2022) for Streptococcus suis, Glaesserella parasuis, Mycoplasma hyorhinis, Actinobacillus suis and Mycoplasma hyosynoviae at Iowa State University Veterinary Diagnostic Laboratory.

BACKGROUND: Accurate measurement of disease associated with endemic bacterial agents in pig populations is challenging due to their commensal ecology, the lack of disease-specific antemortem diagnostic tests, and the polymicrobial nature of swine diagnostic cases. The main objective of this retrospective study was to estimate temporal patterns of agent detection and disease diagnosis for five endemic bacteria that can cause systemic disease in porcine tissue specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2017 to 2022. The study also explored the diagnostic value of specific tissue specimens for disease diagnosis, estimated the frequency of polymicrobial diagnosis, and evaluated the association between phase of pig production and disease diagnosis. RESULTS: S. suis and G. parasuis bronchopneumonia increased on average 6 and 4.3%, while S. suis endocarditis increased by 23% per year, respectively. M. hyorhinis and A. suis associated serositis increased yearly by 4.2 and 12.8%, respectively. A significant upward trend in M. hyorhinis arthritis cases was also observed. In contrast, M. hyosynoviae arthritis cases decreased by 33% average/year. Investigation into the diagnostic value of tissues showed that lungs were the most frequently submitted sample, However, the use of lung for systemic disease diagnosis requires caution due to the commensal nature of these agents in the respiratory system, compared to systemic sites that diagnosticians typically target. This study also explored associations between phase of production and specific diseases caused by each agent, showcasing the role of S. suis arthritis in suckling pigs, meningitis in early nursery and endocarditis in growing pigs, and the role of G. parasuis, A. suis, M. hyorhinis and M. hyosynoviae disease mainly in post-weaning phases. Finally, this study highlighted the high frequency of co-detection and -disease diagnosis with other infectious etiologies, such as PRRSV and IAV, demonstrating that to minimize the health impact of these endemic bacterial agents it is imperative to establish effective viral control programs. CONCLUSIONS: Results from this retrospective study demonstrated significant increases in disease diagnosis for S. suis, G. parasuis, M. hyorhinis, and A. suis, and a significant decrease in detection and disease diagnosis of M. hyosynoviae. High frequencies of interactions between these endemic agents and with viral pathogens was also demonstrated. Consequently, improved control programs are needed to mitigate the adverse effect of these endemic bacterial agents on swine health and wellbeing. This includes improving diagnostic procedures, developing more effective vaccine products, fine-tuning antimicrobial approaches, and managing viral co-infections.

Development of a new multiplex quantitative PCR for the detection of Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae.

Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae are important porcine pathogens responsible for polyserositis, polyarthritis, meningitis, pneumonia, and septicemia causing significant economic losses in the swine industry. A new multiplex quantitative polymerase chain reaction (qPCR) was designed on one hand for the detection of G. parasuis and the virulence marker vtaA to distinguish between highly virulent and non-virulent strains. On the other hand, fluorescent probes were established for the detection and identification of both M. hyorhinis and M. hyosynoviae targeting 16S ribosomal RNA genes. The development of the qPCR was based on reference strains of 15 known serovars of G. parasuis, as well as on the type strains M. hyorhinis ATCC 17981T and M. hyosynoviae NCTC 10167T . The new qPCR was further evaluated using 21 G. parasuis, 26 M. hyorhinis, and 3 M. hyosynoviae field isolates. Moreover, a pilot study including different clinical specimens of 42 diseased pigs was performed. The specificity of the assay was 100% without cross-reactivity or detection of other bacterial swine pathogens. The sensitivity of the new qPCR was demonstrated to be between 11-180 genome equivalents (GE) of DNA for M. hyosynoviae and M. hyorhinis, and 140-1200 GE for G. parasuis and vtaA. The cut-off threshold cycle was found to be at 35. The developed sensitive and specific qPCR assay has the potential to become a useful molecular tool, which could be implemented in veterinary diagnostic laboratories for the detection and identification of G. parasuis, its virulence marker vtaA, M. hyorhinis, and M. hyosynoviae.

Bacterial polyarthritis in post-weaning pigs in a high-density swine breeding area in Italy.

We assessed the bacterial agents found in 8-12-wk-old post-weaning pigs with arthritis. The bodies of 178 post-weaning pigs from 90 farms (average of 2 pigs/farm) with recurrent problems of lameness and swollen joints in a high-density breeding area were submitted for autopsy and sampled for further bacterial investigation. The most common articular gross lesions and histopathologic findings were serofibrinous (95 of 178; 53%) or serous (65 of 178; 37%) arthritis; suppurative lesions were less frequent (18 of 178; 10%). In 133 of 178 (74.7%) cases, a bacterial agent was detected in joints. Mycoplasma hyorhinis was the most common bacterium detected (82 of 133; 61.6%). Haemophilus parasuis and Streptococcus spp. were observed in 27 of 133 (20.3%) and 24 of 133 (18.0%) cases, respectively. Other bacteria in the 113 cases, considered less important, in order of their low frequency, were Mycoplasma spp. (13; 9.8%), Trueperella pyogenes (11; 8.2%), Mycoplasma hyosynoviae (4; 3.0%), Staphylococcus spp. (3; 2.2%), Escherichia coli (2; 1.5%), and Actinobacillus spp. (2; 1.5%). Our results highlight the primary role of M. hyorhinis compared to other microorganisms involved in young pigs with arthritis.

Lameness in fattening pigs - Mycoplasma hyosynoviae, osteochondropathy and reduced dietary phosphorus level as three influencing factors: a case report.

BACKGROUND: Multiple diagnostic procedures, their results and interpretation in a case with severe lameness in fattening pigs are described. It is shown that selected diagnostic steps lead to identification of various risk factors for disease development in the affected herd. One focus of this case report is the prioritization of diagnostic steps to verify the impact of the different conditions, which finally led to the clinical disorder. Assessing a sufficient dietary phosphorus (P) supply and its impact on disease development proved most difficult. The diagnostic approach based on estimated calculation of phosphorus intake is presented in detail. CASE PRESENTATION: On a farrow-to-finishing farm, lameness occurred in pigs with 30-70 kg body weight. Necropsy of three diseased pigs revealed claw lesions and alterations at the knee and elbow joints. Histologic findings were characteristic of osteochondrosis. All pigs were positively tested for Mycoplasma hyosynoviae in affected joints. P values in blood did not indicate a P deficiency, while bone ashing in one of three animals resulted in a level indicating an insufficient mineral supply. Analysis of diet composition revealed a low phosphorus content in two diets, which might have led to a marginal P supply in individuals with high average daily gains with respect to development of bone mass and connective tissue prior to presentation of affected animals. Finally, the impact of dietary factors for disease development could not be evidenced in all submitted animals in this case. CONCLUSIONS: Mycoplasma (M.) hyosynoviae was identified to be an important etiologic factor for disease. Other, non-infectious factors, such as osteochondrosis and claw lesions might have favored development of lameness. In addition, a relevant marginal P supply for pigs was found in a limited time period in a phase of intense growing, but the potential interaction with infection by M. hyosynoviae is unknown. The presented case of severe lameness in fattening pigs revealed that three different influences presumably act in pathogenesis. Focusing only on one factor and ignoring others might be misleading regarding subsequent decision-making for prevention and therapy. Finally, clinical symptoms disappeared after some changes in diet composition and anti-inflammatory treatment of individual animals.

Performance of Commercial Mycoplasma hyopneumoniae Serum Enzyme-Linked Immunosorbent Assays under Experimental and Field Conditions.

Mycoplasma hyopneumoniae is an economically significant pathogen of swine. M. hyopneumoniae serum antibody detection via commercial enzyme-linked immunosorbent assays (ELISAs) is widely used for routine surveillance in commercial swine production systems. Samples from two studies were used to evaluate assay performance. In study 1, 6 commercial M. hyopneumoniae ELISAs were compared using serum samples from 8-week-old cesarean-derived, colostrum-deprived (CDCD) pigs allocated to the following 5 inoculation groups of 10 pigs each: (i) negative control, (ii) Mycoplasma flocculare (strain 27399), (iii) Mycoplasma hyorhinis (strain 38983), (iv) Mycoplasma hyosynoviae (strain 34428), and (v) M. hyopneumoniae (strain 232). Weekly serum and daily oral fluid samples were collected through 56 days postinoculation (dpi). The true status of pigs was established by PCR testing on oral fluids samples over the course of the observation period. Analysis of ELISA performance at various cutoffs found that the manufacturers' recommended cutoffs were diagnostically specific, i.e., produced no false positives, with the exceptions of 2 ELISAs. An analysis based on overall misclassification error rates found that 4 ELISAs performed similarly, although one assay produced more false positives. In study 2, the 3 best-performing ELISAs from study 1 were compared using serum samples generated under field conditions. Ten 8-week-old pigs were intratracheally inoculated with M. hyopneumoniae Matched serum and tracheal samples (to establish the true pig M. hyopneumoniae status) were collected at 7- to 14-day intervals through 98 dpi. Analyses of sensitivity and specificity showed similar performance among these 3 ELISAs. Overall, this study provides an assessment of the performance of current M. hyopneumoniae ELISAs and an understanding of their use in surveillance.

Detection of Mycoplasma hyorhinis and Mycoplasma hyosynoviae in oral fluids and correlation with pig lameness scores.

This study was designed to detect Mycoplasma hyorhinis and M. hyosynoviae in oral fluids and determine their correlation with lameness scores in pigs. Thirty-seven nursery and/or finisher herds were included in this study. Oral fluids were collected by pen. Using species specific real-time PCR M. hyorhinis was detected in 97% of sampled herds, whereas 70% were positive for M. hyosynoviae. Lameness scores were determined for all pigs in each pen where oral fluids were collected. Lameness was identified in 3.9% of pigs across all sampled pens. No correlation was observed between lameness in pigs in a pen and detection of M. hyorhinis in oral fluid samples (p > 0.05), whereas a significant correlation was observed between M. hyosynoviae detection in oral fluids and lameness (p < 0.05). A negative correlation was observed between the proportion of lame pigs in the pen and Ct values for M. hyosynoviae in oral fluids (p < 0.05; r = -0.27). An age-related effect was observed with M. hyosynoviae detection in oral fluids, indicating an increased prevalence of the bacterium in finishers compared to nursery pigs. Under the conditions of this study, M. hyorhinis was frequently detected in oral fluids from nursery and finisher pigs regardless of the clinical presentation of lameness, whereas the detection of M. hyosynoviae varied depending on the age of sample pigs. Our results suggest that oral fluids may not be an informative diagnostic sample for M. hyorhinis associated lameness. However, the association of lameness and M. hyosynoviae detection in oral fluids warrants prospective population-based diagnostic studies.

Transfer of maternal immunity to piglets is involved in early protection against Mycoplasma hyosynoviae infection.

Mycoplasma hyosynoviae causes arthritis in pigs older than 12 weeks. The role of colostrum in protection of piglets against M. hyosynoviae infection is not clear. Our objective was therefore to investigate whether transfer of maternal immunity to piglets was involved in early protection against the infection. Experimental infections were carried out in three groups of weaners receiving different levels of M. hyosynoviae-specific colostrum components; Group NC derived from Mycoplasma free sows and possessed no specific immunity to M. hyosynoviae. Group CAb pigs, siblings of the NC group, received colostrum with M. hyosynoviae-specific antibodies immediately after birth. Group CCE pigs were born and raised by infected sows and presumably had the full set of colostrally transferred factors, including specific antibodies. When 4½ weeks old, all pigs were inoculated intranasally with M. hyosynoviae. The course of infection was measured through clinical observations of lameness, cultivation of M. hyosynoviae from tonsils, blood and synovial fluid and observation for gross pathological lesions in selected joints. Specific immune status in the pigs was evaluated through detection of antibodies by immunoblotting and measurement of M. hyosynoviae-specific T-cell proliferation. The latter analysis may possibly indicate that M. hyosynoviae infection induces a T-cell response. The CCE piglets were significantly protected against development of lameness and pathology, as well as infection with M. hyosynoviae in tonsils, blood and joints, when compared to the two other groups. Raising the CCE pigs in an infected environment until weaning, with carrier sows as mothers, apparently made them resistant to M. hyosynoviae-arthritis when challenge-infected at 4½ weeks of age. More pigs in group NC had M. hyosynoviae related pathological lesions than in group CAb, a difference that was significant for cubital joints when analysed on joint type level. This finding indicates a partially protective effect of passively transferred M. hyosynoviae-specific colostral antibodies upon development of M. hyosynoviae related pathology. Thus, the level of passive immunity transferred from sow to piglet seems to provide, at least partial, protection against development of arthritis. It cannot be ruled out that the CCE pigs, by growing up in an infected environment, have had the chance to establish an active anti-M. hyosynoviae immune response that complements the maternally transferred immune factors. Evident from this study is that the general absence of M. hyosynoviae arthritis in piglets can be ascribed mainly to their immunological status.

Two clinical isolates of Mycoplasma hyosynoviae showed differing pattern of lameness and pathogen detection in experimentally challenged pigs.

Mycoplasma (M.) hyosynoviae is known to colonize and cause disease in growing-finishing pigs. In this study, two clinical isolates of M. hyosynoviae were compared by inoculating cesarean-derived colostrum-deprived and specific-pathogen-free growing pigs. After intranasal or intravenous inoculation, the proportion and distribution pattern of clinical cases was compared in addition to the severity of lameness. Tonsils were found to be the primary site of colonization, while bacteremia was rarely detected prior to the observation of clinical signs. Regardless of the clinical isolate, route of inoculation, or volume of inocula, histopathological alterations and tissue invasion were detected in multiple joints, indicating an apparent lack of specific joint tropism. Acute disease was primarily observed 7 to 10 days post-inoculation. The variability in the severity of synovial microscopic lesions and pathogen detection in joint cavities suggests that the duration of joint infection may influence the diagnostic accuracy. In summary, these findings demonstrate that diagnosis of M. hyosynoviae-associated arthritis can be influenced by the clinical isolate, and provides a study platform to investigate the colonization and virulence potential of field isolates. This approach can be particularly relevant to auxiliate in surveillance and testing of therapeutic and/or vaccine candidates.

Two clinical isolates of Mycoplasma hyosynoviae showed differing pattern of lameness and pathogen detection in experimentally challenged pigs

Mycoplasma (M.) hyosynoviae is known to colonize and cause disease in growing-finishing pigs. In this study, two clinical isolates of M. hyosynoviae were compared by inoculating cesarean-derived colostrum-deprived and specific-pathogen-free growing pigs. After intranasal or intravenous inoculation, the proportion and distribution pattern of clinical cases was compared in addition to the severity of lameness. Tonsils were found to be the primary site of colonization, while bacteremia was rarely detected prior to the observation of clinical signs. Regardless of the clinical isolate, route of inoculation, or volume of inocula, histopathological alterations and tissue invasion were detected in multiple joints, indicating an apparent lack of specific joint tropism. Acute disease was primarily observed 7 to 10 days post-inoculation. The variability in the severity of synovial microscopic lesions and pathogen detection in joint cavities suggests that the duration of joint infection may influence the diagnostic accuracy. In summary, these findings demonstrate that diagnosis of M. hyosynoviae-associated arthritis can be influenced by the clinical isolate, and provides a study platform to investigate the colonization and virulence potential of field isolates. This approach can be particularly relevant to auxiliate in surveillance and testing of therapeutic and/or vaccine candidates.

Comparative genomic analysis of seven Mycoplasma hyosynoviae strains.

Infection with Mycoplasma hyosynoviae can result in debilitating arthritis in pigs, particularly those aged 10 weeks or older. Strategies for controlling this pathogen are becoming increasingly important due to the rise in the number of cases of arthritis that have been attributed to infection in recent years. In order to begin to develop interventions to prevent arthritis caused by M. hyosynoviae, more information regarding the specific proteins and potential virulence factors that its genome encodes was needed. However, the genome of this emerging swine pathogen had not been sequenced previously. In this report, we present a comparative analysis of the genomes of seven strains of M. hyosynoviae isolated from different locations in North America during the years 2010 to 2013. We identified several putative virulence factors that may contribute to the ability of this pathogen to adhere to host cells. Additionally, we discovered several prophage genes present within the genomes of three strains that show significant similarity to MAV1, a phage isolated from the related species, M. arthritidis. We also identified CRISPR-Cas and type III restriction and modification systems present in two strains that may contribute to their ability to defend against phage infection.

Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral, tonsillar, and nasal fluids.

Mycoplasma (M.) hyorhinis and M. hyosynoviae are pathogens known to cause disease in pigs post-weaning. Due to their fastidious nature, there is increased need for culture-independent diagnostic platforms to detect these microorganisms. Therefore, this study was performed to develop and optimize quantitative real-time PCR (qPCR) assays to rapidly detect M. hyorhinis and M. hyosynoviae in pen-based oral fluids as well as nasal and tonsillar fluids as proxies for samples used in swine herd surveillance. Two methods of genomic DNA extraction, automated versus manual, were used to compare diagnostic test performance. A wean-to-finish longitudinal study was also carried out to demonstrate the reproducibility of using pen-based oral fluids. Overall, pen-based oral and tonsillar fluids were more likely to be positive for both types of bacteria whereas only M. hyorhinis was detected in nasal fluids. DNA extraction protocols were shown to significantly influence test result. Although the initial detection time somewhat differed, both organisms were repeatedly detected in the longitudinal study. Overall, this study evaluated two qPCR methods for rapid and specific detection of either mycoplasma. Results from the present investigation can serve as a foundation for future studies to determine the prevalence of the two microorganisms, environmental load, and effectiveness of veterinary interventions for infection control.

Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral, tonsillar, and nasal fluids

Mycoplasma (M.) hyorhinis and M. hyosynoviae are pathogens known to cause disease in pigs post-weaning. Due to their fastidious nature, there is increased need for culture-independent diagnostic platforms to detect these microorganisms. Therefore, this study was performed to develop and optimize quantitative real-time PCR (qPCR) assays to rapidly detect M. hyorhinis and M. hyosynoviae in pen-based oral fluids as well as nasal and tonsillar fluids as proxies for samples used in swine herd surveillance. Two methods of genomic DNA extraction, automated versus manual, were used to compare diagnostic test performance. A wean-to-finish longitudinal study was also carried out to demonstrate the reproducibility of using pen-based oral fluids. Overall, pen-based oral and tonsillar fluids were more likely to be positive for both types of bacteria whereas only M. hyorhinis was detected in nasal fluids. DNA extraction protocols were shown to significantly influence test result. Although the initial detection time somewhat differed, both organisms were repeatedly detected in the longitudinal study. Overall, this study evaluated two qPCR methods for rapid and specific detection of either mycoplasma. Results from the present investigation can serve as a foundation for future studies to determine the prevalence of the two microorganisms, environmental load, and effectiveness of veterinary interventions for infection control.